Cytochromes P450 metabolize arachidonic acid (Aa) to epoxyeicosatrienoic acids in several different tissues including the heart. In humans and rats, the predominant cardiac enzymes responsible for EET production are CYP2J2 and CYP2J3, respectively. Over the past year, we have demonstrated that brief, intermittent periods of cardiac ischemia and reperfusion (preconditioning) protect the rat heart against the functional consequences of a subsequent prolonged period of ischemia and are associated with increased rat heart EET concentrations. Furthermore, micromolar quantities of EETs significantly improve rat cardiac contractility following prolonged, global ischemia suggesting that these endogenous eicosanoids are cardioprotective. In order to study the effects of constitutively increased EETs on cardiac function and on the physiologic response of the heart to ischemia and reperfusion, we have begun to construct transgenic mice that overexpress an active AA epoxygenase in the heart. Constitutive, cardiac-specific overexpression of a soluble, catalytically self-sufficient, active AA epoxygenase (P450 BM-3) will be accomplished using the pGEM-aMHC-SV40 vector which contains the a-myosin heavy chain promotor. We have successfully subcloned the P450 BM-3 cDNA into pGEM-aMHC-SV40 in both a sense and an antisense orientation. The linearized DNAs have been microinjected into pronuclei of single cell fertilized mouse embryos and the eggs have been implanted into pseudopregnant female mice. The offspring are currently being screened for the presence or absence of the P450 BM-3 transgene by PCR and Southern blot analysis. Founder animals will be screened for tissue- specific expression of P450 BM-3 by Northern analysis and protein immunoblotting of mouse tissues. The transgenic mice will then be used to study the role of EETs in cardiac function and during ischemia/preconditioning. We have also recently cloned two new mouse CYp2J subfamily cDNAs (CYP2J5 and CYP2J6), one of which (CYP2J5) is highly and selectively expressed in the heart. Future studies will focus on disruting the respective mouse genes to investigate the effects of reduced EETs on cardiac physiology/pathophysiology.